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  • Title: Mechanism of Pseudomonas aeruginosa Small Protease (PASP), a Corneal Virulence Factor.
    Author: Tang A, Caballero AR, Marquart ME, Bierdeman MA, O'Callaghan RJ.
    Journal: Invest Ophthalmol Vis Sci; 2018 Dec 03; 59(15):5993-6002. PubMed ID: 30572344.
    Abstract:
    PURPOSE: Pseudomonas aeruginosa is the leading cause of contact lens-associated bacterial keratitis. Secreted bacterial proteases have a key role in keratitis, including the P. aeruginosa small protease (PASP), a proven corneal virulence factor. We investigated the mechanism of PASP and its importance to corneal toxicity. METHODS: PASP, a serine protease, was tested for activity on various substrates. The catalytic triad of PASP was sought by bioinformatic analysis and site-directed mutagenesis. All mutant constructs were expressed in a P. aeruginosa PASP-deficient strain; the resulting proteins were purified using ion-exchange, gel filtration, or affinity chromatography; and the proteolytic activity was assessed by gelatin zymography and a fluorometric assay. The purified PASP proteins with single amino acid changes were injected into rabbit corneas to determine their pathological effects. RESULTS: PASP substrates were cleaved at arginine or lysine residues. Alanine substitution of PASP residues Asp-29, His-34, or Ser-47 eliminated protease activity, whereas PASP with substitution for Ser-59 (control) retained activity. Computer modeling and Western blot analysis indicated that formation of a catalytic triad required dimer formation, and zymography demonstrated the protease activity of the homodimer, but not the monomer. PASP with the Ser-47 mutation, but not with the control mutation, lacked corneal toxicity, indicating the importance of protease activity. CONCLUSIONS: PASP is a secreted serine protease that can cleave proteins at arginine or lysine residues and PASP activity requires dimer or larger aggregates to create a functional active site. Most importantly, proteolytic PASP molecules demonstrated highly significant toxicity for the rabbit cornea.
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